**3. Ethnopharmacology**

The isolation and identification of potent compounds from the genus Artemisia, particularly artemisinin and its derivatives using novel drug discovery methods, prompted the Nobel Committee to award the Nobel Prize in Physiology or Medicine in 2015 for its impact on public health [17,18]. This spurred the interest of several researchers to study the phytochemical and pharmacological properties of other species of the genus Artemisia.

Nearly 45 different species of Artemisia grow in India and in the Indian subcontinent and is mainly used as a medicinal plant [8,19]. Ayurveda describes two species *A*. *absinthium* and *A. maritima,* popularly known as Mugwort that vary slightly in their qualities and actions as shown in Table 1.

**Table 1.** Ayurveda describes two species *A. absinthium* and *A*. *maritima* that vary slightly in their qualities and actions. Both plants popularly known as Mugwort are revered in Ayurveda for their anti-infectious and insecticidal (*krimighna*) properties [20].


In the Ayurvedic system of medicine, the term '*prabhava*' refers to the 'instinct intelligence' of a plant in eliciting a wide range of medicinal effects [21,22]. *A*. *absinthium* and *A. maritima* are revered, owing to their *prabhava* and are recommended in Ayurveda for infections, inflammation, skin and liver diseases, respiratory conditions, neurological conditions and as an insecticidal (*krimighna*) [6,8].

The pharmacological actions and properties of the various Artemisia species from several geographic locations are listed in Table 2. Basically, the plant has been used as an anti-malarial, anti-spasmodic, anti-inflammatory, febrifuge, cardiac stimulant, anthelmintic, headaches, dyspepsia, liver and kidney tonic, to improve memory, for digestive and respiratory issues and as a hypertensive and anticoagulant.


**Table 2.** The pharmacological actions and properties of a subset of Artemisia species.

The wide variety of actions stems from the fact that these various species of Artemisia possess high content of alkaloids, lactones, flavonoids, phenols, quinines, tannins and terpenoids all of which play a role in the growth of the plant or provide protection from pathogens or predators [6,9,44,45].

#### **4. In Vitro and In Vivo Studies**

We review some of the recent in vivo and in vitro studies of various extracts and formulations of Artemisia. The research studies utilized aqueous, methanol, chloroform or acetone extracts, essential oils or oil based extracts or dried powders of various species of Artemisia. The studies were performed on bacterial, viral or fungal cultures, cultured cells or animal models with limited studies on humans.

In the light of the COVID-19 pandemic, some species of Artemisia including but not limited to *A. annua*, *A. absinthium*, *A. vulgaris*, *A. maritima* and *A. indhana* are receiving greater attention from researchers as they hold grea<sup>t</sup> potential for their powerful antiinfectious, antiviral and anti-inflammatory activities [6,12,46–48]. Recent studies are now pointing to the exciting roles of artemisinin and its derivatives (ARTs) as potential drug candidates against SARS-CoV-2 owing to their potent antiviral and anti-inflammatory properties.

#### *4.1. Anti-Carcinogenic Activity*

Various species of the Artemisia plant have been shown to suppress the growth of numerous cancer cell lines including leukemia, colon cancer, renal cell carcinoma and breast cancer cells [28,49,50]. Phytochemical analysis of the various extracts revealed the presence of coumarins, flavonoids, anthocyanins, cardiac glycosides and tannins. These phytochemicals and their derivatives exhibit growth inhibitory properties through multiple actions including blocking angiogenesis, triggering apoptosis or cell cycle arrest and disrupting cell migration [51–53]. Researchers are now focusing their efforts on ARTs that appear to be broad-spectrum antitumor agents based on their efficacy and safety [54,55].

In a randomized, double-blind, placebo-controlled pilot trial involving 23 subjects, the anticancer effect and tolerability of oral artesunate in colorectal cancer (CRC) was determined. The primary outcome measure was the proportion of tumor cells undergoing apoptosis. Despite the fact that it was a small study size with variability in quantitating immunohistochemical markers, the results clearly indicated selective cytotoxicity of oral artesunate.

In addition to the above mentioned study, other clinical trials involving patients with solid tumors including colorectal carcinoma, breast cancer, hepatocellular carcinoma and lung cancer have been completed with encouraging results. In all these studies, ARTs inhibited growth of solid tumors with no evident toxicity and with a low incidence of adverse effects thus highlighting their role as promising anti-cancer agents [54,56,57].

#### *4.2. Anti-Oxidant Activity*

The phytochemicals and their derivatives, extracts and essential oils derived from the Artemisia plant have a unique property of being reactive oxygen species (ROS) modulators. In some cases they exhibit strong antioxidant and radical scavenging activity against hydroxyl ion and hydrogen peroxide and display excellent protective effect by strengthening the antioxidant defense system and lowering the generation of ROS [6,58].

In other situations, especially involving cancer cells, ARTs triggered ROS production leading to mitochondrial dysfunction and autophagy of leukemia cell lines. ARTs-induced ROS production triggered apoptosis in various tumor cell lines studies, including neuroblastoma, glioblastoma, T-cell lymphoma and breast cancer cells [54]. In studies using mouse models of cancer, ARTs induced ROS production leading to the inhibition of growth of ovarian cancer [54].

The mechanism of action of ARTs involves binding to ferrous iron (e.g., heme) and triggering the generation of ROS, which results in cytostatic or cytotoxic effects. The production of ROS can also trigger cellular damage through the peroxidation of membrane lipids, activation of pro-apoptotic pathways or creating genomic and mitochondrial DNA instability [59]. Thus, the ROS modulating properties exhibited by the various phytochemicals isolated from different species of Artemisia highlight the importance of exploring the therapeutic uses of these compounds in pathological conditions that feature oxidative stress.

#### *4.3. Anti-Bacterial and Anti-Parasitic Activity*

The plant extracts and compounds obtained from Artemisia species have been shown to be powerful inhibitors of bacteria and parasites [9]. Mechanistic studies demonstrate the bactericidal properties of some of these phytochemicals against Gram-negative or Gram-positive bacteria involving the destruction of the bacterial membrane [6,28,60,61]. Notable among the phytochemicals is ARTs that represent a new class of antibacterial drugs [9,14,15].

ARTs also possess potent antimalarial properties and are effective against both asexual and sexual parasite stages. In several clinical trials involving both ARTs and quinine, ARTs outperformed quinine in terms of mean parasite clearance time, fever clearance time, coma resolution times and incidence of adverse effects [14–16,62]. Artemisinin-based therapies are now recommended due to the resistance displayed by bacteria and parasites to quinoline drugs.

#### *4.4. Anti-Fibrotic Effects*

In addition to the above mentioned pharmacological properties, ARTs are also known for their anti-fibrotic effects [29,63,64]. The role of ARTs in blocking the development or progression of fibrotic phenotypes has been studied in animal models of pulmonary fibrosis, renal fibrosis, hepatic fibrosis, and other types of tissue fibrosis suggesting the potential utility of these compounds as anti-fibrotic agents. The effects of ARTs against profibrotic processes include induction of apoptosis, inhibition of proliferation, blocking differentiation of tissue-specific myofibroblast precursors or preventing the accumulation of tissue myofibroblasts that provoke tissue fibrosis [6,63]. In addition, ARTs block the expression of extracellular matrix (ECM) genes and pro-fibrotic genes in myofibroblasts thereby antagonizing cellular processes that promote accumulation of fibrotic tissue. ARTs also inhibit angiogenesis either through direct effects on endothelial cells or indirectly by downregulating pro-angiogenic gene expression in angiogenesis-supporting non-endothelial cells. With its anti-fibrotic role in disease models across several species and multiple tissues involving diverse mechanisms, artemisinin-based therapeutics for treatment of fibrotic diseases may prove efficacious in humans [64].

#### *4.5. Role in Neurodegeneration*

Extracts of several Artemisia species exhibit neuroprotective effects against focal ischemia-reperfusion-induced cerebral injury, microglial cytotoxicity and glutamate excitotoxicity [65]. Furthermore, Artemisia protects neurons against mitochondrial potential loss, attenuates reactive oxygen species and protects neurons against H2O2-induced death by upregulating the Nrf2 pathway [66]. ARTs improve learning and memory in mouse models of Alzheimer's disease mice by blocking Aβ25-35-induced increase in the levels of inflammatory cytokines IL-1β, IL-6 and TNF-α and by restoring the autophagic flux and promoting the clearance of Aβ fibrils [67,68].

Recently, three different subtypes of Alzheimer's disease (AD) have been described [69]. The type-3 AD classified as infectious or Krimi (ayurveda classification of AD) is the result of exposure to virus or biotoxins, such as mycotoxins, and features chronic inflammation [69,70]. Owing to their powerful antiviral and anti-inflammatory properties, ARTs may serve as excellent drug candidates for type-3 AD.

#### *4.6. Anti-Inflammatory Activity*

Artemisia species exhibit powerful anti-inflammatory effects. Several sesquiterpenes derived from Artemisia and their derivatives including artemisinin, artesunate, dihydroarteannuin, artemisolide, eupatilin, scoparone, capillarisin and scopoletin have received special attention due to their role in blocking inflammation. Using animal models, ARTs were found to be effective in treating inflammatory conditions including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and allergic disorders [71].

Some of the anti-inflammatory mechanisms include: (1) inhibition of the iNOS and COX-2 pathways; (2) suppression of ERK and NF-κB signaling; (3) inhibition of pathogenic T cell activation; (4) suppressing B cells activation and antibody production; and (5) inhibition of Akt phosphorylation and IκB degradation through the PI3K/Akt signaling pathway downstream of TNFα [72–75]. Thus, the varied mechanisms through which these phytochemicals derived from Artemisia exhibit their anti-inflammatory effects warrant investigation into their role as therapeutic candidates for inflammatory conditions and autoimmune disorders.
